1
CHAPTER 5-1
TARDIGRADE SURVIVAL
Figure 1. Dactylobiotus on the green alga Spirogyra. Photo by Yuuji Tsukii.
Tardigrades – Water Bears
Tardigrades (tardus = slow, gradus = step, or slow
walkers), also known as water bears or moss piglets, are
close relatives of the arthropods (Garey et al. 1996, 1999;
Giribet et al. 1996).
Water bears resemble small bears (0.1-1 mm),
complete with claws, but a few too many legs (4 pairs)
(Figure 1). They are either armored (Heterotardigrada) or
unarmored (Eutardigrada). The aquatic ones are usually a
translucent white, whereas the terrestrial ones are often
colored. Each of the eight legs has claws, which, when
combined with their slow gait, makes them look very much
like miniature polar bears with some extra legs. The very
common Macrobiotus hufelandi (Figure 2) lumbers along
at a maximum of 17.7 cm h-1 (Ramazzotti & Maucci in
Mach, The Water Bear). Tardigrades are just the right size
to move among the bryophyte leaves, they lumber along
slowly like bears, and they are downright cute!
Tardigrades can be found in marine, aquatic, and
terrestrial habitats. On land they frequently live in
association with bryophytes (Figure 3; Figure 4) and
lichens (Miheleie 1967; Mehlen 1969; Utsugi 1984;
Meininger et al. 1985; Mancardi 1988; Szymanska 1994;
Bertolani & Rebecchi 1996; Tarter et al. 1996; Miller
1997; Jarez Jaimes et al. 2002; Boeckner et al. 2006;
Bartels et al. 2009; Meyer & Hinton 2009; Rossi et al.
2009; Simmons et al. 2009).
Figure 2. Macrobiotus hufelandi, a common tardigrade that
is among those inhabiting mosses. Photo by Paul Bartels.
These terrestrial tardigrades depend on the water drops
that adhere to mosses and liverworts (Hingley 1993) and
are therefore often termed limnoterrestrial (living in
terrestrial habitats, but requiring a water film). Aquatic
bryophytes can also house tardigrades (Hallas 1975;
Kinchin 1987b, 1988; Steiner 1994a, b), as do the algae.
However, of the ~1000 tardigrades reviewed by Guidetti
and Bertolani (2005) and Garey et al. (2008), only 62 were
2
Chapter 5-1: Tardigrade Survival
truly aquatic. The others depend on water associated with
the interstitial spaces of terrestrial algae, lichens,
bryophytes, and leaf litter. Water bears are found in
habitats from hot springs to layers under the ice (in
cryoconite holes in glaciers) and occupy every continent of
the world.
Figure 3. This tardigrade resided among the leaves of the
moss Hypopterygium arbuscula. Photo by Filipe Osorio.
Figure 5. Echiniscus species (E. testudo occurs almost
exclusively on bryophytes) seems to be unresponsive to moisture
changes. Photo by Martin Mach.
Suitability of Bryophytes as Habitat
Figure 4. Hypopterygium arbuscula, a known bryophyte
habitat for tardigrades in Chile. Photo by Juan Larrain through
Creative Commons.
Despite their cosmopolitan distribution (Romano
2003), broad habitat requirements, and relative visibility
(compared to protozoa, for example), the tardigrades
remain poorly known. As late as 1985, Hidalgo and
Coombs reported that 16 states in the USA had no records
of tardigrades. Species not previously described are easily
discovered by those who know where to look for them.
Most of the terrestrial tardigrades are bryophyte
inhabitants (Nelson 1991a). These terrestrial bryophyte
taxa have a life span ranging from 3-4 months (Franceschi
et al. 1962-1963), 3-7 months for Macrobiotus hufelandi
(Figure 2; Morgan 1977), up to about 3 months for roof
moss dwelling Echiniscus testudo (Morgan 1977), to about
2 years (Altiero & Rebecchi 2001) of active life (not
counting dormant periods). The bryophyte-inhabiting taxa
are more common in temperate and polar zones than in the
tropics (Nelson 1991a). Some, as for example Echiniscus
testudo (Figure 5), live almost exclusively on bryophytes
(Corbet & Lan 1974).
The importance of bryophytes as a tardigrade habitat is
evident by the number of publications on "moss" tardigrade
fauna: Miheleie 1967; Hallas 1975; Pilato & Sperlinga
1975; Morgan 1976; Bruegmann 1977; Morgan 1977;
Maucci 1978, 1980; Bertolani 1983, 2001; Binda 1984;
Utsugi 1984; Meininger et al. 1985; Hofmann 1987;
Hofmann & Eichelberg 1987; Kinchin 1987a, b, 1988,
1994; Meininger & Spatt 1988; Mancardi 1988; Bertolani
et al. 1990; Tarter & Nelson 1990; Kathman & Cross
1991; Nelson 1991a, b; Utsugi & Ohyama 1991; Moon et
al. 1994; Szymanska 1994; Miller & Heatwole 1995;
Adkins & Nelson 1996; Tarter et al. 1996; Hooie &
Davison 2001; Guidetti & Jönsson 2002; Jönsson 2003;
Meyer et al. 2003; Hooie: Tardigrade Diversity), to name a
few. It appears that when tardigrade lovers want to collect
a lot of them, they collect bryophytes and lichens – or just
bryophytes (generally lumped into "mosses").
Unfortunately, the authors rarely name the bryophytes
from which their prizes were extracted. However, some
evidence suggests that little specificity exists for bryophyte
species, and lichens are as suitable as bryophytes, with no
apparent differences in tardigrade species (Meyer & Hinton
2007). I have to wonder, however, why reports on
tardigrades from liverworts are so scant (Figure 6).
Perhaps it is just as suggested to me by Łukasz Kaczmarek,
that most zoologists do not understand the differences
between mosses and liverworts. (Neither do my students
when they begin looking at them.)
Ramazzotti and Maucci (1983) considered mosses
suitable habitat based on three needs of the
limnoterrestrial tardigrades:
1. a structure that allows sufficient oxygen diffusion
2. the ability to undergo alternate periods of wetting and
drying resulting from solar radiation and wind
3. a medium that contains sufficient food.
Chapter 5-1: Tardigrade Survival
3
Figure 7. Encalypta streptocarpa, a tardigrade habitat that
can be difficult to navigate when it is fully hydrated. Photo by
Michael Lüth.
Figure 6. Tardigrades on the lower sides of leaves of a leafy
liverwort. Photo by Łukasz Kaczmarek and Łukasz Michalczyk.
Based on these criteria, bryophytes are particularly
good habitats for tardigrades in several ways (Ramazzotti
& Maucci 1983; Claps & Rossi 1984; Adkins & Nelson
1996). Their structure permits sufficient oxygen diffusion,
both in aquatic and terrestrial habitats.
Bryophytes
experience drying, which they do slowly, permitting the
tardigrades likewise to dry slowly, and both have a
tolerance to dehydration that permits them to survive
adverse conditions (Kinchin 1994). Furthermore, the
tardigrades have a prolonged life span when it is
interrupted by such a dormancy period. And bryophytes
contain food items such as algae, protozoa, and nematodes,
as well as the bryophytes themselves, sufficient for the
tardigrades. Most likely, the small chambers among the
bryophyte branches also afford protection from larger
would-be predators. And when fragments of bryophytes
disperse, they may carry tardigrades with them.
But bryophytes do pose their problems for the tiny
tardigrades. These animals are quite light weight, so
imagine their struggle to control their movements when
they encounter fully hydrated bryophytes with a continuous
bath of water surrounding them. Greven and Schüttler
(2001) observed these slow-moving creatures (Macrobiotus
sp., Echiniscus testudo) on Encalypta streptocarpa [=E.
contorta] (Figure 7) when the bryophyte was fully
hydrated. The poor bears could barely move and had
difficulty maintaining the direction of their movements in
the water. They could easily become dislodged by
rainwater unless they are able to nestle in a leaf axil or
other protected niche. And that is often a good place to
look for them.
On the other hand, Polytrichastrum [=Polytrichum]
formosum (Figure 8) did not sustain a continuous water
film and the tardigrades seemed also unable to move in this
"dry" habitat (Greven & Schüttler 2001). Rather, they
seemed confined to the leaf axils, where water collected.
As water receded, the animals ceased movement and
formed a tun (protective dormant stage of tardigrade that is
altered both chemically and physically) right there!
Perhaps tardigrades were the inspiration for the Rip Van
Winkle story.
Figure 8. Polytrichastrum formosum, a moss that does not
maintain a water film and is thus a poor tardigrade habitat. Photo
by Des Callahan.
Moisture seems to be the greatest determinant of the
species distribution among bryophytes. Their richness
among epiphytic bryophytes in the Cincinnati, Ohio, USA
area was greatest in areas of high humidity (Meininger et
al. 1985). Hofmann and Eichelberg (1987) found that the
tardigrades lacked correlation with bryophyte species but
that their distribution could be predicted by the degree of
moisture they prefer. It is therefore not surprising that
some bryophytes housed no tardigrades.
Tardigrades in association with roads along the Alaska
pipeline demonstrate a moisture relationship (Meininger &
Spatt 1988). Dust resulting from gravel roads associated
with the pipeline alters the habitat for both mosses and
tardigrades. Those tardigrades living among mosses near
roads were species adapted to xeric habitats. These species
typically fed on fungi and algae, whereas those farther from
the road were more likely to be omnivores or carnivores,
presumably because they had more freedom to move about.
Adaptations of Tardigrades
One might ask if these bryophyte-dwelling creatures
have any special adaptations that permit them to live where
they do. Their greatest adaptation is that they live in a
habitat that permits them to dry slowly and go into a
dormant state, as we will discuss shortly – a kind of
behavioral/physiological adaptation. Like the insects, they
have chitin, in this case in the innermost layer of the
4
Chapter 5-1: Tardigrade Survival
cuticle. The chitinous armor of some terrestrial tardigrades
(heterotardigrades) may slow drying and offer protection
from damage while dry. Of course their small size is
essential for living in the miniature world of bryophytes.
And their claws (Figure 9 - Figure 11) may permit them to
clamber about more easily among the leaves and branches
of the bryophytes.
Their light weight facilitates their dispersal. For many,
the stylets permit them to suck the contents out of
bryophyte cells, among other things. Their bodies are
flexible, permitting them to nestle in leaf axils or move in
small spaces. But most of these as adaptations to the
bryophyte habitat are speculation. There have been no tests
to determine if any of these traits actually increases their
survival in bryophytes compared to other habitats. Some
very interesting experiments could be designed.
Let's examine one of the bryophyte-dwelling
tardigrades as an example of potential adaptations. Martin
Mach (The Water Bear) found Cornechiniscus cornutus
(Figure 12) among bryophytes on a mountain top in
Hungary. This cute little bear has two horns on its head
(Figure 13) and a nice salmon color. But it is slow and
clumsy, out-classed by the faster-moving and more
abundant Ramazzottius oberhaeuseri (Figure 19). Do such
ornamentations as horns and hairs help to reduce predation
in this habitat? Is that an advantage to offset the slower
movement? Does the bright color protect the water bear
from UV damage, especially while it is dry?
Figure 9. Claws on four of the eight legs of Echiniscoides
sigismundi (a tidal zone species). Photo by Martin Mach.
Figure 12.
Mach.
Cornechiniscus cornutus.
Photo by Martin
Survival of Hazardous Conditions
Figure 10. Claws of a tardigrade that is most likely
Cornechiniscus cornutus (a bryophyte-dweller). Photo by Martin
Mach.
The biggest hazard a bryophyte imposes on a
tardigrade is intermittent desiccation. But in addition to
that desiccation, the organism may be subjected to high or
low temperatures, low oxygen conditions, and UV light for
prolonged periods. With little ability to move elsewhere, it
needs some other type of protection.
Figure 13. Cornechiniscus cornutus showing one of its two
head horns. Photo by Martin Mach.
Figure 11. Claws of Echiniscus sp., a genus with many
bryophyte-dwelling species. Photo by Martin Mach.
Aquatic organisms rarely need to be concerned with
desiccation. However, if an animal is to survive among
terrestrial bryophytes, it must be prepared for drying when
the bryophyte dries out, and many of the tardigrade habitats
are in dry places, including cryptogamic crusts
(assemblages of Cyanobcteria, algae, lichens, & mosses) in
Chapter 5-1: Tardigrade Survival
5
the prairie and desert and epiphytes on trees. These bring
with them the very hazards mentioned above – UV light in
the absence of water for protection, and extremes in
temperature. And the fleshy body must be hydrated for
oxygen to enter it.
Physical Adaptations
The soft-bodied tardigrades appear to have few
structural adaptations to survive drought. Some, like
Echiniscus, have long hairs (Figure 14 - Figure 15), but the
hairs are so few that one can hardly imagine they are of any
help to reduce water loss or protect the dry animal.
Hmmm...What might their function be?
Figure 16. Cornechiniscus cornutus showing armor. Photo
by Martin Mach.
Figure 17. Echiniscus showing a type of armor. Photo by
Martin Mach.
Figure 14. Echiniscus trisetosus, illustrating the sparse but
long hairs. Photo by Łukasz Michalczyk and Łukasz Kaczmarek.
Figure 15. Two of the long hairs of Echiniscus. Photo by
Martin Mach.
Others may have a bit more protection. Some
bryophyte-dwelling species such as Cornechiniscus
cornutus (Figure 16) and some members of the genera
Echiniscus (Figure 17 - Figure 18) and Ramazzottius
(Figure 19 - Figure 20) (and others) have "armor" on their
bodies that is somewhat leathery. I am aware of no studies
that demonstrate the ability of the armor to reduce water
loss, but it would appear to be a good possibility. Other
possible advantages of this armor-like cuticle may include
protection from fungi and other pathogens and some kinds
of predators, particularly while in cryptobiosis, and it most
likely would afford limited UV protection. How little we
know!
Figure 18. Echiniscus posterior dorsal side showing a type
of armor. Photo by Martin Mach.
It is possible that the wonderful colors of some
tardigrades (Figure 21 - Figure 22) are adaptations against
UV damage to DNA during prolonged periods in a
cryptobiotic state. Terrestrial tardigrades come in green,
brown, yellow, orange, pink, red, purple, or black, whereas
aquatic ones are white (Hebert 2008). Such pigmentation
advantages have been demonstrated in bryophytes
(Martínez Abaigar & Olivera 2007) and copepods (Byron
1982), so it is reasonable to expect them to serve similar
functions in tardigrades, particularly in those more open
habitats such as cryptogamic crusts. It would be an
interesting study to examine the relationship of color with
habitat in tardigrades. I am aware of no such study.
6
Chapter 5-1: Tardigrade Survival
Figure 21. Adult Echiniscus, demonstrating one of the bright
colors found in tardigrades. Photo by Martin Mach.
Figure 19. Ramazzottius oberhaeuseri, a tardigrade with
armor. Photo by Martin Mach.
Figure 22. Cornechiniscus cornutus, a bryophyte-dwelling
"horned" species that exhibits brilliant colors that could afford UV
protection. Photo by Martin Mach.
Figure 20. Armor on Ramazzottius oberhaeuseri. Photo by
Martin Mach.
Cryptobiosis
Albert Szent-Gyorgyi, a 20th Century Hungarian
biochemist, once stated "Water is life's mater and matrix,
mother and medium. There is no life without water." In their
cryptobiotic state, tardigrades come close to disproving that
statement.
Literally meaning "hidden life," cryptobiosis is a state
of suspended animation in which the organism is able to
survive unfavorable conditions without expending much
energy. During that state, the organism does not feed,
reproduction stops, and metabolism is extremely reduced
and may possibly even cease. For the limnoterrestrial
(living in water films on land) tardigrade, it appears to be
an essential part of survival and life, and it stops the aging
clock.
Despite the apparent absence of structural adaptations,
desiccated tardigrades, like their mossy habitats, have great
survival capabilities. They have two forms of dormancy:
cryptobiosis and encystment (Guidetti et al. 2006). The
cryptobiosis of tardigrades is exhibited in several forms:
anhydrobiosis (induced by loss of water)
cryobiosis (induced by declining temperatures)
anoxybiosis (induced by insufficient oxygen)
osmobiosis (induced by loss of water due to higher
external salt concentrations) (Bertolani et al. 2004).
To be active, tardigrades must stay in a water film in
order to breathe (Bordenstein: Tardigrades). But in a
cryptobiotic state, as discussed below, tardigrades can
survive not only desiccation, but temperatures as low as
0.05K (-272.95ºC) for 20 hours or -200ºC for 20 months
(Miller 1997). They have even survived 151ºC for a few
minutes (Lindahl & Balser 1999). They become active
again after living with 0% hydration (Lindahl & Balser
1999). This desiccated dormant state also permits them to
survive pressures of 6000 atmospheres (Seki & Toyoshima
1998), i.e. six times the pressure of the deepest part of the
oceans! Yet they can also survive the vacuum and UV
radiation of space (Jönsson et al. 2008), a feat not known
for any other animal. The tardigrades will be the ones to
survive when everything else is deceased.
Chapter 5-1: Tardigrade Survival
7
The ability of tardigrades to undergo cryptobiosis is
more widely known than their encystment behavior. True
cryptobiotic states are survived as a tun.
Tun Formation
When they undergo desiccation, the tardigrades form a
tun (Figure 23 - Figure 33) (Lindahl & Balser 1999). The
tun is a barrel-shaped, dry, dormant tardigrade. Tuns are
formed in the process of entering true cryptobiosis, i.e., in
anhydrobiosis, osmobiosis, and cryobiosis, but not in
anoxybiosis. Although the stimulus differs among these,
each ultimately involves the loss of free water.
Figure 26. Tun of Hypsibius sp. Photo by Martin Mach.
Figure 23. Tun of Ramazzottius oberhaeuseri. Photo by
Martin Mach.
Figure 27. Tun of Echiniscus. Photo by Martin Mach.
Figure 24. Tardigrade tun – water bear in a state of
anhydrobiosis. Photo by Janice Glime.
Figure 28. Tun of Echiniscus on moss leaf. Photo by Martin
Mach.
Figure 25. Tardigrade tun – water bear in a state of
anhydrobiosis. Note the buccal apparatus (resembles a tuning
fork on left end). Photo by Janice Glime.
Figure 29. Multiple tuns of Echiniscus on a single moss leaf.
Photo by Martin Mach.
8
Chapter 5-1: Tardigrade Survival
Figure 30. Tun of Echiniscus on moss leaf. Photo by Martin
Mach.
Figure 31. Tun of Echiniscus on a moss leaf. Photo by
Martin Mach.
This tun is a little ball in which the tardigrade can
survive 0% relative humidity! However, it only requires a
reduction to 70-95% humidity to trigger the tun formation,
a resting form in a cryptobiotic state in which the
tardigrade appears to be dead (Crowe 1972). During tun
formation, loss of free and bound water is greater than 95%
(Bertolani et al. 2004). The body folds and the appendages
are withdrawn (Lindahl & Balser 1999). Wax is extruded
onto the surface and most likely reduces water loss (Wright
1988a, b). Those tardigrades with the most variability in
the thickness of this cuticle, making them more pliable, are
those able to have the greatest surface area reduction when
they form tuns (Wright 1988a, 1989). The thin areas would
permit greater infolding. Lipids of the inner cuticle are
thickest in the species that are best able to tolerate rapid
drying. Despite the waxy protection, the water content is
reduced to less than 1% (Lindahl & Balser 1999) and the
tun becomes shrivelled and wrinkled (Hingley 1993).
Echiniscus testudo, an armored tardigrade, has much
thicker dorsal (back) plates, apparently compensating for
its limited ability to reduce surface area as it is drying
(Wright 1988a, 1989). The tardigrade bodies synthesize
cell protectants such as trehalose, glycerol, and heat
shock proteins that contribute to their successful recovery
from the tun state.
Tun formation is essential to tardigrade survival under
desiccating conditions. For Paramacrobiotus areolatus,
and probably most tardigrades, if the humidity is low
(<70%) or anoxic (lacking oxygen) during its desiccation,
it is unable to form a tun and cannot be revived (Crowe
1972). It must have sufficient energy (requiring oxygen),
hydration, and time to enter the tun stage.
They revive (Figure 34) almost as quickly as a moss
when water returns (Crowe & Higgins 1967), in as little as
4 minutes (Hingley 1993), or several hours, depending on
how long they have been dehydrated (Lindahl & Balser
1999). One marine tardigrade has been induced to alternate
between a cryptobiotic state and activity on a 6-hour cycle.
Figure 32. Tun of Echiniscus. Photo by Martin Mach.
Figure 34. Echiniscus sp. rehydrated after four years of
desiccation. Photo by Martin Mach.
Dangers in a Tun
Figure 33. Tun of Echiniscus. Photo by Martin Mach.
One concern that comes to mind is the possible
damage that could happen to these organisms while in the
tun stage. I am reminded of the frozen frogs and toads
during the winter. They are very susceptible to physical
damage if they are disturbed. I would think an animal such
as the amphibians hiding under a rock or clump of moss
Chapter 5-1: Tardigrade Survival
would experience no more physical abuse than the tiny
tardigrade among the moss leaves. Ice crystals could poke
holes in cells, larger animals could eat them, or they could
get knocked off into a hole where conditions were not
favorable to their maintenance and survival. I have to
wonder just what dangers these dormant organisms do face,
and how many actually survive these in the wild to become
once again active. It seems we currently have no idea.
Certain dangers include cell degradation and DNA
damage. As the tardigrades exist longer and longer, they
accumulate cell degradation and DNA damage (Rebecchi et
al. 2009), ultimately accumulating too much for successful
repair. Hence, the tun does not completely protect them,
and their chances of survival decrease with time.
9
Within Richtersius coronifer (Figure 37), large
individuals were less likely to survive cryptobiosis than
medium-sized ones (Figure 38); reproductive state had no
effect (Jönsson & Rebecchi 2002). Better energetic
conditions increased survival. Jönsson and Rebecchi
suggested that larger organisms had greater energy
constraints when entering and leaving anhydrobiosis,
decreasing survival rate.
Effects of Size
Jönsson et al. (2001) found that size influenced
survival of cryptobiotic tardigrades, but that direction of
influence differed among species.
The common
Ramazzottius oberhaeuseri (300 µm length; Figure 35) had
a much higher survival rate (66%) (Figure 36) than did
Richtersius coronifer (40%) (up to 1 mm length; Figure 37).
Ramazzottius oberhaeuseri has a high ability to retain
water, perhaps contributing to an advantage for larger
individuals with lower surface area to volume ratio.
Figure 37. Richtersius coronifer, clinging to an algal cell.
Photo by Martin Mach.
Figure 35. Ramazzottius oberhaeuseri. Photo by Martin
Mach.
Figure 38. Probability of survival from anhydrobiosis for
large and medium-sized Richtersius coronifer as a function of
storage cell size. Probability is based on the predicted values
from a logistic regression model, using buccal tube length,
category, storage cell size, and interaction between the last two
categories. Redrawn from Jönsson & Rebecchi 2002, in Bertolani
et al. 2004.
Figure 36. Comparison of survival during encystment for
Richtersius coronifer and Ramazzottius oberhaeuseri from Italy
and Sweden. Vertical line represents standard error. Redrawn
from Bertolani et al. 2004, based on Jönsson et al. 2001.
Jönsson and Rebecchi (2002) likewise found that
medium-sized tardigrades had a better chance of survival
than did large ones in Richtersius coronifer. Large storage
cell size was an important parameter to predict greater
survival in the large tardigrades (Figure 38).
Reuner et al. (2010) described the storage cells as freefloating cells in Milnesium tardigradum (Figure 39),
10
Chapter 5-1: Tardigrade Survival
Paramacrobiotus tonollii, and Macrobiotus sapiens that
apparently store and release energy as glycogen, protein,
and fat. These stores provide energy during cryptobiosis.
Storage cell size did not relate to body size, except that the
largest tardigrade, Milnesium tardigradum, also had the
largest storage cells. After seven days of anhydrobiosis
(tun stage resulting from desiccation), this species had
decreased cell size, but the other two species did not. Food
sources used in the study did not seem to affect cell size.
Figure 40. Grimmia pulvinata, a moss that can support
tardigrade communities on roofs. Photo by Michael Lüth.
Figure 39. Milnesium tardigradum, a large tardigrade.
Photo by Yuuji Tsukii.
Longevity
Tardigrades are often credited with century-long
survival in a cryptobiotic state. This is due to the report
that one herbarium specimen of a moss housed a tardigrade
that began cellular activity after 120 years of being dry in
the herbarium (Franceschi 1948; Brusca & Brusca 1990;
Jönsson and Bertolani 2001)! But, sadly, this record has
been called into question, and the tardigrade never fully
recovered. At the very best, even this faint degree of
survival is probably a rare occurrence (see Jönsson &
Bertolani 2001). Jönsson and Bertolani (2001) reviewed
the evidence and considered that ten years is a more
realistic estimate of survival time in a cryptobiotic state.
Rebecchi et al. (2008) decided to test this claim of
longevity further, using five species of tardigrades from
lichens. They collected wet lichens with active tardigrades
and permitted them to dry in the ambient conditions of the
lab. Among these, Ramazzottius oberhaeuseri, Echiniscus
testudo, and E. trisetosus (species that also occur on
bryophytes) were sufficiently abundant to permit statistical
conclusions. At the beginning of the experiment 91% of R.
oberhaeuseri and 72% of Echiniscus spp. were active.
Ramazzottius oberhaeuseri survived up to 1604 days,
whereas Echiniscus spp. lived only 1085 days.
To test the longevity of tuns vs eggs under
anhydrobiosis, Guidetti and Jönsson (2002) examined 63
different moss samples from stored collections, ranging in
anhydrobiotic state 9 - 138 years. Eggs survived longer
than dry adults (tuns), with those of Ramazzottius
oberhaeuseri surviving nine years. Much more work is
needed to determine what factors account for such
differences in survivorship and how it relates to individual
species and habitats. The ability to survive unfavorable
conditions permits the tardigrades to live in such places as
Grimmia pulvinata tufts (Figure 40) on house roofs (Corbet
& Lan 1974) or among branches of the epiphyte
Orthotrichum cupulatum (Figure 41) (Jönsson et al. 2001).
Figure 41. Orthotrichum cupulatum, an acrocarpous moss
that provides habitats for tardigrades. Photo by Michael Lüth.
Like the rotifers, tardigrades suspend their aging clock
while they are dormant (Hengherr et al 2008). Milnesium
tardigradum that was subjected to alternating periods of
drying and activity exhibited similar longevity of active
periods to that of animals of the species that had not
experienced dry periods.
Ramazzotti and Maucci (1983) estimated that
freshwater species such as those of Hypsibius (Figure 42)
and Macrobiotus (Figure 43) live about 1-2 years.
Terrestrial bryophyte-inhabiting species of the same genera
live much longer, averaging 4–12 years. This extended life
is due largely to their periods of cryptobiosis, during which
the biological clock stops.
Figure 42. Hypsibius convergens, a common bryophyte
inhabitant. Photo by Paul Bartels.
Chapter 5-1: Tardigrade Survival
11
Trehalose is not a cure-all for desiccation effects in
tardigrades. High temperatures and high humidity may
lead to destruction of trehalose (Rebecchi et al. 2009). In
other cases, or in consort, oxidative damage may occur.
Using Paramacrobiotus richtersi as an experimental
organism, Rebecchi et al. demonstrated that DNA changes
can occur during desiccation. Neumann et al. (2009)
likewise demonstrated a slight increase in DNA damage
during drying, but they also found that DNA damage
increased with duration of anhydrobiosis. Furthermore,
high temperatures and relative humidity have negative
effects on both survival and time to recover after
rehydration, with effects increasing with duration of
exposure. One reason for this is that damages are not
repaired during anhydrobiosis and therefore accumulate
with time.
Figure 43. Macrobiotus marlenae, a terrestrial species
known from mosses on rock. Photo by Martin Mach.
Dangers and Protective Mechanisms
One contributing factor in their survival of drying is
the ability of tardigrades to alter their cell membranes
(Brave New Biosphere 1999). They replace the water in
the cell membranes with sugar, thus preventing radiation
from causing ionization. Like the nematodes and rotifers,
some tardigrades prepare for desiccation by producing
disaccharide sugars, including trehaloses (Bordenstein:
Tardigrades; Westh & Ramløv 1991). Disaccharides like
trehalose and sucrose, as well as glycerol, are used as
membrane protectants by metazoans such as tardigrades,
whereas plants typically use oligosaccharides such as
stachyose and raffinose (Wright 2001).
This water replacement by sugars also protects
invertebrates during freezing because crystallization cannot
occur (Brave New Biosphere 1999). The accumulation of
trehalose of 0.1-2.3% of dry weight occurred within 5-7
hours during desiccation in Richtersius coronifer (Westh &
Ramløv 1991). This accumulation was reversed within 6
hours upon rehydration. Both water loss and sugar
replacement prevent the rupture of the cell membrane that
would result in death. But trehalose has multiple properties
that help to stabilize desiccated cells (Table 1).
Table 1. Properties of trehalose that benefit dehydrating
cells. From Watanabe 2006.
Non-reducing activity
Low tendency to crystallize
Stable glass formation
High vitrification temperature
High ability of water replacement
Structuring activity of intracellular water with HSP
Stabilization of dry membranes
Antioxidant activity of protein and fatty acids
Free-radical scavenger
Nevertheless, tardigrades accumulate trehalose at the
low end of the scale for anhydrobiotic organisms – about
2% (Watanabe 2006). This lower level in tardigrades and
absence of trehalose in rotifers is coupled with their ability
to enter anhydrobiosis within one hour, whereas organisms
with larger accumulations (up to 40%) can take at least two
days.
Anhydrobiosis
The most common of the cryptobiotic states is
anhydrobiosis (state of dormancy brought on by
dehydration). In their state of anhydrobiosis, tardigrades
can remain inactive during unfavorable conditions such as
prolonged dryness (Kinchin 1987b). Anhydrobiosis is
usually restricted to animals less than 1 mm in length
(Watanabe 2006). Hence, some invertebrates are only able
to enter this state during early developmental stages.
Tardigrades and rotifers, being less than 1 mm when fully
developed, are able to do so at any developmental stage.
In order to survive anhydrobiosis, tardigrades must dry
very slowly (Hingley 1993; Collins & Bateman 2001). To
form the tun, they must retract their head, legs, and hind
end, forming a rounded tun, thus reducing surface area. In
this state of anabiosis, they are able to withstand extremes
of temperature and desiccation.
Nevertheless, water
arouses them in as little as four minutes.
It appears that continuously hydrated conditions may
be detrimental to the survival of tardigrades (Jönsson
2007). Using bryophyte populations from Island Öland,
Sweden, Jönsson subjected the tardigrades to two
treatments of 6-month duration over an 18-month period.
These experimental treatments increased hydration,
decreased hydration, or remained as controls. The total
population was significantly smaller (barely so) under
increased hydration. But effects were not the same for all
tardigrades.
Richtersius coronifer (Figure 37) and
Echiniscus spiniger failed to respond to the treatment,
whereas Milnesium tardigradum declined under increased
hydration. But even Richtersius coronifer experienced
reduction in the density of eggs (Figure 44 - Figure 45)
Hydration did not
under the watering treatment.
significantly increase density in any of the tardigrades.
This adds further support to the idea that periods of
dormancy (cryptobiosis) are necessary to increase
longevity of the tardigrade. This would, in turn, increase
variability of conditions, offering an array of conditions for
reproduction.
Richtersius coronifer can increase its survival rate by
forming aggregates, a mechanism barely known for
tardigrades but common in nematodes (Ivarsson & Jönsson
2004). The clustering reduces exposed surface area and
thus slows drying. It is possible that this is used more in
tardigrades than is realized; its use among bryophyte fauna
is as yet unknown.
12
Chapter 5-1: Tardigrade Survival
Balser 1999). Tardigrades are very sensitive to changes in
oxygen tension, and prolonged reduction of oxygen leads to
osmoregulatory failure.
Anoxybiosis is not a true state of cryptobiosis and does
not involve tun formation (Figure 46. Unlike true
cryptobiosis, anoxybiosis involves the uptake of water.
The lack of oxygen results in the inability to control
osmosis, causing water to enter the cells in excess. The
animals become turgid, immobile, and retain fully extended
bodies that are perfectly bilaterally symmetrical (Figure
47). Even animals in a molt can enter anoxybiosis (Figure
48).
Figure 44. Egg of Richtersius coronifer. Photo by Martin
Mach.
Figure 46. Macrobiotus hufelandi male in anoxybiotic state,
showing lack of tun formation. Photo by Martin Mach.
Figure 45. Macrobiotus magdalenae egg showing the highly
decorated nature that is typical of eggs laid free from the exuvia
(shed body shell). In this state it can survive as well as in a tun.
Photo by Łukasz Kaczmarek and Łukasz Michalczyk.
Figure 47. Tardigrade showing anoxybiosis, where water
has entered through the cuticle by osmosis and caused swelling
and turgidity. Note the extended legs and perfectly symmetrical
body. The animal cannot move in this state. Photo by Martin
Mach.
The cryptobiotic state of anhydrobiosis has a
significant impact on the ecological role of the tardigrades.
It affects their role in the food chain, their ability to
disperse, and their survival through a longer period of time
(see reviews by Pilato 1979; Wright et al. 1992; Kinchin
1994).
Osmobiosis
Osmobiosis is a special case of cryptobiosis that
permits some species to tolerate high salinity and to form a
tun (Lindahl & Balser 1999). It is initiated when the
animal experiences an external salt concentration that is
higher than that inside the organism. However, for
tardigrades, while possible, osmobiosis is typically not
necessary as most tardigrades already have a high salt
tolerance.
Anoxybiosis
Anoxybiosis is another special case where the
tardigrade has the ability to survive low oxygen (Lindahl &
Figure 48. Tardigrade induced into anoxybiosis during its
molt. Photo by Martin Mach.
Revival to normal state (Figure 49) relates to the
duration of the dormant state. However, the success of that
recovery is controversial (Wright et al. 1992), with some
researchers finding that they can survive for only 3-4 days
(Crowe 1975) and others finding survival of Echiniscoides
Chapter 5-1: Tardigrade Survival
(a tidal zone genus) up to six months in closed vials
(Kristensen & Hallas 1980).
Cryobiosis
Cryobiosis is another special case of cryptobiosis that
results when the temperature decreases and the water in the
cells has frozen (Wikipedia:
Cryptobiosis 2009).
Molecular mobility stops (Wikipedia: Cryptobiosis 2009),
permitting the tardigrades to survive very low temperatures
(Westh et al. 1991; Westh & Kristensen 1992; Ramløv &
Westh 1992; Sømme 1996; McInnes & Pugh 1998). They
do this by actually freezing, but the freezing is ordered
(Lindahl & Balser 1999) and the result once again is a tun.
Figure 49. This tardigrade was caught by low oxygen during
molt and entered anoxybiosis. Here it has recovered and is
moving within the swollen cuticle to complete its molt. Photo by
Martin Mach.
Tardigrades often experience wide temperature
fluctuations while in an active state. In particular, they can
be subjected to subzero temperatures. Their ability to
tolerate these sub-zero conditions requires either tolerance
of freezing body water or having a mechanism to lower the
freezing point. Hengherr et al. (2009) subjected nine
species from polar, temperate, and tropical regions to
cooling by 9, 7, 5, 3, and 1ºC h-1 down to -30ºC, then
returning them to ambient temperature at 10ºC h-1.
Survival was better at fast and slow cooling rates, with low
survival rates at intermediate cooling rates. Hengherr et al.
suggested that this relationship may indicate a physical
effect during fast cooling and possible synthesis of
cryoprotectants during slow cooling. The increased
survival with slower cooling indicates that tardigrades
protect their cellular structure from freezing injury without
altering their freezing temperature.
At least some protection seems to be accomplished by
using ice-nucleating proteins in the body fluids (Westh et
al. 1991). Such proteins serve as centers for crystal
formation, a technique used to make snow for ski hills.
This cryoprotective mechanism permits tardigrades to
survive rapid freezing and thawing cycles such as those
experienced in the Arctic and Antarctic. Usually this type
of protection means that the nucleating centers are small,
permitting only small crystals to form, consequently
reducing damage to the cell membranes.
13
The ice-nucleating activity in the body fluid from
Richtersius coronifer is reduced by 50% following ca
7x103 times dilution (Westh et al. 1991). Heating to
temperatures above 68°C induces an abrupt decrease in the
activity, suggesting that the nucleators are proteinaceous.
Westh and Kristensen (1992) examined Richtersius
coronifer and Bertolanius [=Amphibolus] nebulosus and
compared their cryoprotective strategies.
Richtersius
coronifer lives in drought-resistant mosses and overwinters
in a frozen or dry state (cryptobiosis). Bertolanius
nebulosus, on the other hand, lives among moist mosses
and algae and spends its winter frozen in a cyst or as eggs.
Both species can supercool to as low as -7ºC. But these
two species have distinctly different heat stability, resulting
from differences in ice-nucleating proteins. In both cases,
ice formation is rapid, but crystallization most likely stops
within a minute of nucleation. This protects the cells from
damage caused by large, sharp crystals. Nevertheless, ice
constitutes 80-90% of the body water.
Winter
acclimatization of R. coronifer results in a 10% lower ice
formation than summer acclimatization. The thaw point
was unaffected by winter vs summer, suggesting that there
is no accumulation of low molecular weight cryoprotective
substances.
Despite their seeming indestructibility, not all
tardigrade individuals fare well at low temperatures, and
some species fare better than others. Bertolani et al. (2004)
demonstrated this for three species of tardigrades (Figure
50). Ramazzottius oberhaeuseri seems to be almost
indestructible down to -80ºC, whereas Hypsibius dujardini
had only 20% survival at that temperature. In fact, it had
less than 80% survival at -9ºC.
Figure 50. Comparison of survival of three bryophytedwelling tardigrades subjected to sub-zero temperatures. Redrawn
from Bertolani et al. 2004.
Diapause (Encystment)
Tardigrades are especially endowed with the
physiological ability to survive. They are among the few
organisms that can use both anhydrobiosis and diapause
(encystment) as a means of dormancy to survive
unfavorable conditions (Guidetti et al. 2008). Diapause is
common among aquatic tardigrades, but there are some
terrestrial species that experience diapause (Westh &
Kristensen 1992; Nelson 2002). Whereas cryptobiosis is
well studied, the role of diapause (encystment) is not well
known in tardigrades. It appears that it is not an essential
part of the life cycle – only a means to survive some
unfavorable conditions.
Węglarska (1957) found that Dactylobiotus dispar
(Figure 51 - Figure 54) was induced to encyst by
14
Chapter 5-1: Tardigrade Survival
environmental conditions that gradually became worse.
Interestingly, when there was a rapid change to poor
conditions, this tardigrade went into anoxybiosis. When a
tardigrade is about to encyst, it ingests large amounts of
food that is stored in the body cavity cells (Nelson 1991a).
The remaining material in the gut is defecated.
they produce two or three new cuticles. In Bertolanius
[=Amphibolus] volubilis, the new cuticle is similar to that
found on the non-encysted organisms, whereas in
Dactylobiotus parthenogeneticus the ultrastructure of the
new cuticle differs. The tardigrade retracts within the
cuticle (Nelson 1991a).
Figure 51. Dactylobiotus sp. Photo by Yuuji Tsukii.
Figure 54. Egg of Dactylobiotus dispar. Photo by Martin
Mach.
Figure 52. Dactylobiotus dispar. Photo by Martin Mach.
Tardigrade encystment is known for only a few
species, although it may be more widespread than is
currently known. There are at least three types of cysts
(Guidetti et al. 2006). Bertolanius volubilis has two types
(Figure 55); Dactylobiotus parthenogeneticus (Figure 56 Figure 58) exhibits only one. Having two types of cysts in
the same species seems to be a terrestrial character
(Bertolani et al. 2004). Type 2 cysts have an additional
layer of cuticle compared to type 1 cysts. Although only a
few species have been described, it appears that a type 1
cyst never shows a modified buccal-pharyngeal apparatus,
whereas a type 2 cyst does.
Figure 53. Eggs of Dactylobiotus dispar. Photo by Martin
Mach.
Encystment is more complex than tun formation
(Bertolani et al. 2004). The cysts are ovoid and are
composed of a series of cuticles that surround the sleeping
animal (Guidetti et al. 2006). They are described as
resembling an onion or a Matrioshka Russian doll.
During encystment, new cuticular structures are
synthesized (Guidetti et al. 2006). Encystment starts with
the discharge of the sclerified portions of the buccalpharyngeal apparatus without the loss of cuticle. Rather,
Figure 55. Upper: Type 1 cyst. Lower: Type 2 cyst
(surrounded by several layers of cuticle), both of Bertolanius
volubilis. Photos by Roberto Bertolani in Bertolani et al. 2004,
reproduced with permission.
Chapter 5-1: Tardigrade Survival
15
Using Bertolanius volubilis from the mosses
Racomitrium sudeticum (Figure 59) and R. elongatum
(Figure 60) on sandstone in the Northern Apennines of
Italy, Guidetti et al. (2008) examined the factors involved
in the inducement of diapause. They learned that in B.
volubilis the type of diapause cysts produced in April
differed from those produced in November. The April
cysts are produced during a warm season, whereas the
other type is present during the cold season. Temperature
is responsible for induction, maintenance, and termination
of the cyst. Both exogenous (temperature) and endogenous
factors serve as stimuli.
Figure 56. Dactylobiotus sp., a member of a genus with only
one type of diapause. Photo by Yuuji Tsukii.
Figure 59. Racomitrium sudeticum, where Bertolanius
volubilis in the Northern Apennines of Italy undergoes diapause,
forming spring cysts that differ from winter cysts. Photo by
Michael Lüth.
Figure 57. Dactylobiotus sp., a tardigrade with only one type
of diapause cyst. Photo by Martin Mach.
Figure 60. Racomitrium elongatum, another moss habitat in
the Northern Apennines of Italy where Bertolanius volubilis
makes different cysts in spring and winter. Photo by Michael
Lüth.
Figure 58. Dactylobiotus sp. cyst.
Photo by Roberto
Bertolani in Bertolani et al. 2004, reproduced with permission.
Conditions that cause emergence from the cysts are not
understood. Unlike those in an anhydrobiotic state, the
encysted tardigrades are not drought-resistant. Nor can
they withstand high temperatures, because they have
continuous water content. Nevertheless, the cysts can
survive in nature for more than a year on their food
reserves (Westh & Kristensen 1992).
Eggs
As already noted, eggs can provide a long-lasting
escape from unfavorable conditions. At least some
tardigrades can produce both subitaneous (non-resting)
and resting eggs (Bertolani et al. 2004). Altiero et al.
(2009) examined the eggs of Paramacrobiotus richtersi
and found that the percentage of hatching was high (7593%), but that four different patterns were discernible.
Subitaneous eggs hatched in 30-40 days. Delayed hatching
eggs hatched in 41-62 days. Some eggs required 90 days
or more if the culture was wet and 13% of these (diapause
resting eggs) required a dry period followed by
rehydration. The remainder (87% of this last >90-day
category) never hatched. They considered this variable
hatching time to be a form of bet-hedging.
16
Chapter 5-1: Tardigrade Survival
Eggs that are laid externally are typically ornamented
(Figure 61 - Figure 62) (Nelson 1991a). These may be laid
singly or in groups.
Figure 63. Milnesium tardigradum, a bryophyte dweller
whose younger stages are the most susceptible to desiccation.
Photo by Yuuji Tsukii.
Figure 61. Egg of a tardigrade, a stage that helps it survive
desiccation. Photo by Martin Mach.
Migration?
Anhydrobiosis is not the only strategy available to
organisms to escape drying conditions. Some organisms
migrate to deeper levels of the moss or soil to escape
drought. However, it appears that this option might not be
available to the slow-moving tardigrades.
Nelson and Adkins (2001) examined this depth
relationship in cushions of the moss Schistidium rivulare
[=Schistidium alpicola] (Figure 64). They found that
among five species, only one (Echiniscus viridissimus) was
more frequent in the top layer, regardless of the wet or dry
condition of the moss. (Hmmm... Could the green that
gives it its name indicate it has a photosynthetic symbiont
that requires light, or just a penchant for green food?)
Figure 62. Macrobiotus szeptyckii egg showing the highly
decorated surface of eggs laid free from the exuvia. Photo by
Łukasz Kaczmarek and Łukasz Michalczyk.
Developmental Stages
Schill and Fritz (2008) examined the effects of
humidity levels (10, 20, 31, 40, 54, 59, 72, 81%) on five
different developmental stages of Milnesium tardigradum
(Figure 63). They determined that the younger stages were
more sensitive to desiccation. During the first three days of
development, low humidity decreased the hatching rate
following rehydration. When embryos were subjected to
low humidity, development was delayed and they
experienced a reduction in hatching rates following
rehydration. At least in this case, further development
affords greater survival of drought.
Figure 64. Schistidium rivulare, a moss where excessive
hydration can cause death to its tardigrade inhabitants. Photo by
Michael Lüth.
None of the species appeared to use migration as a
means to escape reduction in moisture. Nelson and Adkins
(2001) speculated that for tardigrade inhabitants of xeric
mosses, there was no advantage to migration. Rather, they
stayed put and went into a state of anhydrobiosis in both
upper and lower layers.
Chapter 5-1: Tardigrade Survival
Summary
Tardigrades (water bears) are common in both
aquatic and terrestrial bryophytes. The land dwellers
require a water film and thus are called
limnoterrestrial tardigrades. Despite their worldwide
distribution, they are not well known.
The bryophyte habitat offers sufficient oxygen,
wetting and drying, sufficient food, a dispersal vehicle,
and protection.
Moisture is probably the most
important factor in their distribution. Species of
bryophytes do not seem to affect the types of
tardigrades species.
Tardigrades are adapted to the bryophyte habitat by
their small size, stylets that permit sucking contents
from bryophyte cells, flexible bodies, and a very
responsive life cycle. Colored pigments in some may
offer UV protection, especially during dry periods.
Tardigrades can encyst or go into a cryptobiotic state
as a tun. Cysts may differ between summer and winter.
Tardigrades must dry slowly to survive the cryptobiotic
state. While in it, they are resistant to high and low
temperature extremes, absence of water, extreme
pressure, vacuum, and radiation. Anhydrobiosis is
induced by diminishing hydration; cryobiosis is
induced by low temperatures near 0ºC; osmobiosis is
induced by a change in salinity; anoxybiosis is induced
by low oxygen. Tardigrades form trehaloses that
protect the cell membranes while dehydrated or at low
temperatures. They typically can survive about 10
years in the tun, but one specimen resumed
physiological activity after 120 years on a herbarium
moss specimen, then died. Nevertheless, DNA damage
accumulates during cryptobiosis; survival seems to be
based on DNA repair. Furthermore, high temperatures
and high humidity destroy trehalose.
Another means of long-term survival is by
producing resistant eggs. Variable hatching times may
provide a form of bet-hedging in some species.
Acknowledgments
Like all of my chapters, this one is really the product
of the efforts of many biologists. Roberto Bertolani
provided an invaluable update to the tardigrade taxonomic
names, offered several suggestions on the text to provide
clarification or correct errors, and obtained permission to
use his published photographs from the Journal of
Limnology. Paul Davison and Des Callahan have been
helpful in providing suggestions and offering images.
Filipe Osorio has sent me images several times, thinking of
this project even when I was not soliciting help. Martin
Mach and Yuuji Tsukii have given permission to use their
many images that illustrate the species and life cycle
stages. Martin Mach's website has been invaluable.
Łukasz Kaczmarek has provided me with references,
images, contact information, and many valuable comments
on early stages of the manuscript. Marty Janners and
Eileen Dumire provided me with the views of two novices
in the readability of the text. Thank you to Michael Lüth
for permission to use his many images and to all those who
have contributed their images to Wikimedia Commons and
other public domain sites for all to use. I fear I have
17
forgotten some who have helped – I have worked on this
chapter for too many years.
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